Vasoconstrictive and antibacterial combination treatment for rosacea

ABSTRACT

The invention relates to the field of medicine, specifically to the field of treatment of rosacea. The invention relates to a novel composition and a novel kit of parts, both comprising a vasoconstrictive compound and a compound specifically targeting a bacterial cell, preferably a gram positive bacterial cell. The invention further relates to said composition and/or kit of parts for medical use, preferably for treating an individual suffering from rosacea.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a US national phase under 35 U.S.C. § 371 ofinternational application PCT/EP2017/075407, filed Oct. 5, 2017, whichclaims priority to international application PCT/EP2016/074036, filedOct. 7, 2016, which are incorporated herein by reference in theirentirety.

FIELD OF THE INVENTION

The invention relates to the field of medicine, specifically to thefield of treatment of rosacea. The invention relates to a novelcomposition and a novel kit of parts, both comprising a vasoconstrictivecompound and a compound specifically targeting a bacterial cell,preferably a gram positive bacterial cell. The invention further relatesto said composition and/or kit of parts for medical use, preferably fortreating an individual suffering from rosacea.

BACKGROUND OF THE INVENTION

Rosacea is a chronic inflammatory condition of the facial skin affectingthe blood vessels and pilosebaceous units. Rosacea is more common inpersons of northern and western European descent with a fair complexion,but it can affect skin of any color. Although symptoms may wax and waneduring the short term, rosacea can progress with time.

Patients usually have complaints of flushing, blushing, and sensitiveskin. They may even be unaware of these symptoms prior to diagnosis, buta variety of triggers, or factors that induce or exacerbate rosacea,exist. Rosacea is manifested as erythematous flushing, blushing,telangiectasia, papules, and pustules affecting the central third of theface. In areas of long-standing disease, yellow-orange plaques (phymas)can develop, resulting from sebaceous hyperplasia, most commonly on thenose (rhinophyma). The red papules, pustules, and telangiectasia appearin the same distribution, albeit it with a lower frequency, in Asiansand Hispanics; however, because of the pigmentation, they may not appearas erythematous. African-Americans generally do not have red papules anderythema; instead, they have the granulomatous form of rosacea. Manyexperts report that rosacea can occur in areas other than the face. Inerythemato-telangiectatic rosacea (ETR), one may observe macular rednessof the ears, the lateral facial contours, the neck, the upper portion ofthe chest, and the scalp. These extra facial manifestations in ETR areuncommon and are usually seen only in areas affected by flushing and bychronic sun damage. Acneiform lesions have been observed on the centralpart of the chest and on the scalp, the neck and, occasionally, thelimbs.

Possible treatments of rosacea include azelaic acid, topicalmetronidazole, and oral tetracyclines, in particular minocycline anddoxycycline. Other topical treatments include topical clindamycin,subantimicrobial-dose doxycycline and sulfur products. Azithromycin andcontrolled-release minocycline are other possible options for treatingrosacea. For an extensive and comprehensive review on rosacea, see Culpand Scheinfeld, 2009; Pharmacy and Therapeutics, Vol 34 No. 1, page38-45, which is herein incorporated by reference [8].

Rosacea is thus a complex inflammatory skin disorder and no precisetreatment algorithm has become standard; treatment remains empirical.Treatment with e.g. topical and/or oral antibiotics is rather based ontheir symptomatic effect than on their antibacterial efficacy.Symptomatic treatment of erythematous flares, stinging and burning withthe alpha₁-agonist oxymetazoline has been reported [9].

Accordingly, there is a need for improved treatment of rosacea extendingmere symptomatic and empirical treatment.

FIGURE LEGENDS

FIG. 1. Total symptom scores of rosacea during monotherapy andcombination therapy with a vasoconstrictor and Staphefekt™.

The symptom scores are expressed relative to the total score withouttherapy.

-   -   No therapy: White bars    -   Vasoconstrictor therapy: Shaded bar (left)    -   Staphefekt™ therapy: Shaded bar (right)    -   Combination therapy of vasoconstrictor and Staphefekt™: Black        bar

DESCRIPTION OF THE INVENTION

An inappropriate innate immune response against environmental triggersis considered to play a major role in the pathogenesis of rosacea [1,2]. Antigens of microbes are considered to act as an environmentaltrigger by stimulating Toll Like Receptor 2 (TLR 2) that is found to beover-present in the rosacea skin, resulting in inflammatory effects[3-5]. Staphylococcus aureus (S. aureus) is known for its ability tostimulate TLR 2 and its presence could thus attribute to inflammation inrosacea [2, 6, 7].

The inventors have established that a combination of an antibacterialagent and a vasoconstrictor results in effective treatment of rosacea,telangiectasia, erythema and/or flushing. Without being bound to theory,it is believed that the bacterial trigger for inflammation is suppressedby the antibacterial agent, resulting in less inflammation-relatedvasodilatation, in turn allowing a lower need for a vasoconstrictivecompound. The combined effect of said combination of compound has asurprising synergistic effect. Accordingly, in a first aspect, theinvention provides for a novel composition comprising a first and asecond compound, wherein said first compound is a vasoconstrictor andsaid second compound is a compound specifically targeting a bacterialcell, preferably a gram positive bacterial cell. Preferably, said grampositive bacterial cell is a Staphylococcus, more preferably aStaphylococcus aureus. Preferably, said composition is a medicamentpreferably for use in the treatment of rosacea, more preferably for usein the treatment of telangiectasia, erythema and/or flushing, preferablytelangiectasia, erythema and/or flushing associated with inflammationinduced vasodilatation, as further detailed herein. The inventionprovides for a novel composition comprising both a vasoconstrictivecompound and a compound specifically targeting a bacterial cell,preferably a gram positive bacterial cell, preferably a Staphylococcusaureus, which combats most, if not all, of the disadvantages of usingeither an effective dosage regime of a vasoconstrictive compound and/ora conventional topical or oral agent alone or in combination andprovides a unexpected synergy. In comparison to the use ofvasoconstrictive compound alone, a combination according to theinvention decreases the risk and/or is more effective in treatingrosacea, more preferably telangiectasia, erythema and/or flushing,preferably telangiectasia, erythema and/or flushing associated withinflammation induced vasodilatation. Furthermore, in comparison to theuse of a vasoconstrictor alone, a composition according to the inventionmay be as effective as using a vasoconstrictor alone while making use ofa substantially lower dosage and/or a shorter administration regimenresulting in a shorter exposure time of the vasoconstrictor therebyreducing possible side-effects.

In comparison to the use of a vasoconstrictor in combination withconventional antibiotics and/or conventional antibiotics alone, thecomposition of the invention selectively targets a bacterial cell,preferably a gram positive bacterial cell, preferably a Staphylococcus,more preferably a Staphylococcus aureus, without affecting surroundingcommensal and/or beneficial microflora. In addition, the risk ofdeveloping resistance against antibiotics is diminished or at leastreduced since lower amounts of antibiotics or even no antibiotics at allare used.

An agent that specifically targets a gram positive bacterial cellpreferably is an agent that shows at least 2, 5, 10, 50 or 100 timeshigher lytic activity towards a gram positive bacterial cell as comparedto a gram negative bacterial cell. Preferably, an agent thatspecifically targets a gram positive bacterial cell is an agent thatdoes not affect a gram negative bacterial cell in a concentration thatis affective in lysing a gram positive bacterial cell. An agent thatspecifically targets a Staphylococcus bacterial cell preferably is anagent that shows at least 2, 5, 10, 50 or 100 times higher lyticactivity towards a Staphylococcus bacterial cell as compared to anon-Staphylococcus bacterial cell. Preferably, an agent thatspecifically targets a Staphylococcus bacterial cell is an agent thatdoes not affect a non-Staphylococcus bacterial cell in a concentrationthat is effective in lysing a Staphylococcus bacterial cell. An agentthat specifically targets a Staphylococcus aureus bacterial cellpreferably is an agent that shows at least 2, 5, 10, 50 or 100 timeshigher lytic activity towards a Staphylococcus aureus bacterial cell ascompared to a non-Staphylococcus aureus bacterial cell. Preferably, anagent that specifically targets a Staphylococcus aureus bacterial cellis an agent that does not affect a non-Staphylococcus aureus bacterialcell in a concentration that is effective in lysing a Staphylococcusaureus bacterial cell. Lytic activity is preferably assessed by aturbidity assay as described elsewhere herein.

Preferably, the invention provides a composition comprising a first anda second compound, wherein said first compound is an a vasoconstrictivecompound and said second compound is a compound specifically targeting abacterial cell, preferably a gram positive bacterial cell, and whereinsaid second compound comprises at least one cell wall binding domainspecifically binding the peptidoglycan cell wall of said bacterial cell,preferably gram positive bacterial cell. A cell wall-binding domain ofthe invention is defined as an element, preferably a polypeptide withinsaid second compound that directs said second compound to the bacterialwall of a bacterial cell.

A cell wall-binding domain encompassed within the invention may be anycell wall-binding domain known by the person skilled in the art.Preferably, a cell wall-binding domain of the invention is an element,preferably a polypeptide within said second compound, that directs saidsecond compound to the peptidoglycan cell wall of a gram-positivebacterial cell, preferably the peptidoglycan cell wall of aStaphylococcus bacterial cell, more preferably the peptidoglycan cellwall of a Staphylococcus aureus bacterial cell.

Preferably, the invention provides a composition comprising a first anda second compound, wherein said first compound is a vasoconstrictivecompound and said second compound is a compound specifically targeting abacterial cell, preferably a gram positive bacterial cell, and whereinsaid second compound comprises at least one cell wall binding domainspecifically binding the peptidoglycan cell wall of Staphylococcus, morepreferably, a Staphylococcus aureus.

Binding of a domain to the peptidoglycan cell wall of Staphylococcusgenera may be assessed using assays well known to the person skilled inthe art. In a preferred embodiment, an immunohistochemical techniqueand/or a gene fusion technique resulting in labelled constructs are usedfor assessing specific binding of compounds such as peptides,polypeptides, proteins or bacteriophages to the peptidoglycan cell wallof Staphylococcus genera. Quantification methods of signals used in theabove mentioned immunohistochemical or fusion techniques are well knownin the art.

In one embodiment, Staphylococcus peptidoglycan cell wall-binding isquantified using a fluorescent fusion construct comprising a cellwall-domain of interest. Such a cell wall-binding assay is described indetail by Loessner et al (Molecular Microbiology 2002, 44(2): 335-349).In this assay a solution comprising said fluorescent fusion construct ora negative control, preferably Green Fluorescent Protein (GFP), issubjected to Staphylococcus cells, preferably S. aureus cells, morepreferably S. aureus BB255 for an indicated time period where after thecells are sedimented by centrifugation together with the boundfluorescent fusion constructs. The fluorescent signal of theStaphylococcus cells exposed to a fluorescent fusion constructsubtracted by the fluorescence signal of the Staphylococcus cellsexposed to a negative control, preferably GFP, is a measure for cellbinding as meant in this disclosure. Preferably, within the context ofthe invention, a domain is said to bind the peptidoglycan cell wall ofStaphylococcus genera when using this assay an increase in fluorescentsignal of the sedimented cells above the negative control as definedherein is detected. Preferably, the invention relates to a cellwall-binding domain which exhibits binding as defined herein of at least50, 60, 70, 80, 90 or 100, 150 or 200% of peptidoglycan cellwall-binding of S. aureus bacteriophage (I2638a endolysin (Ply2638endolysin defined by SEQ ID NO: 2) preferably encoded by SEQ ID NO: 1.Preferably, a fusion construct as represented by SEQ ID NO: 95 andencoded by SEQ ID NO: 96 serves as a positive control in this assay. Anoverview of all sequences included and their SEQ ID NO is given in table1.

Preferably, the invention provides for a composition comprising a firstand a second compound, wherein said first compound is a vasoconstrictivecompound and said second compound is a compound specifically targeting abacterial cell, preferably a gram positive bacterial cell, and whereinsaid second compound comprises at least one cell wall binding domainthat originates from or is a homologue of a Staphylococcus phageendolysin, preferably said Staphylococcus phage endolysin is selectedfrom, but not limited to, S. aureus bacteriophage Φ2638a endolysin, S.aureus bacteriophage Φ11 endolysin, S. aureus bacteriophage ΦTwortendolysin, S. haemolyticus JCSC1435, S. aureus Phage K endolysin, S.warneri phage WMY endolysin, S. aureus phage NM3 endolysin and S. aureus80alpha endolysin.

Also preferred is a cell wall binding domain originating from or ahomologue of S. simulans lysostaphin (represented by SEQ ID NO: 76,preferably encoded by SEQ ID NO: 75). A known homologue of S. simulanslysostaphin having cell wall binding properties is S. capitis ALE-1enzyme.

Preferably, said cell wall binding domain has at least 80% identity toany of SEQ ID NO: 4, 6 or 8 and/or wherein said one or more enzymaticactive domains has at least 80% identity to any of SEQ ID NO: 10, 12,14, 16, 18, 98 or 100. A preferred cell wall-binding domain of theinvention is a cell wall-binding domain having at least 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%identity with the cell wall binding domain of S. simulans lysostaphindefined herein by SEQ ID NO: 4 and preferably encoded by SEQ ID NO: 3.Also preferred is a cell wall-binding domain isolated from a nativeStaphylococcus bacteriophage endolysin. Also preferred is a cellwall-binding domain of the invention that has at least 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%identity with the cell wall-binding domain of S. aureus bacteriophageΦ2638a endolysin defined herein by SEQ ID NO: 6 and preferably encodedby SEQ ID NO: 5. Also preferred is a cell wall-binding domain isolatedfrom a native Staphylococcus aureus phage phiNM3 endolysin. Preferably,a cell wall-binding domain of the invention has at least 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%identity with the cell wall-binding domain of S. aureus phage phiNM3endolysin defined herein by SEQ ID NO: 8 and preferably encoded by SEQID NO: 7.

Preferably, the invention provides for a composition comprising a firstand a second compound, wherein said first compound is a vasoconstrictivecompound and said second compound is a compound specifically targeting abacterial cell, preferably a gram positive bacterial cell, wherein saidsecond compound comprises one or more enzymatic active domainsexhibiting target bond specificity. ‘An enzymatic active domain’ isdefined herein is a domain having lytic activity, preferably exhibitingpeptidoglycan hydrolase activity. Lytic activity can be assessed bymethods well known by the person skilled in the art. In an embodiment,lytic activity is assessed spectrophotometrically by measuring the dropin turbidity of substrate cell suspensions. Turbidity is assessed bymeasuring optical density at a wavelength of 595 nm, typically a cultureas turbid when it exhibits an optical density of at least 0.3 OD at awavelength of 595 nm. Preferably, lytic activity is assessedspectrophotometrically measuring the drop in turbidity of a S. aureussuspension, wherein turbidity is quantified by measuring OD₅₉₅spectrophotometrically (Libra S22, Biochrom). More preferably, 200 nMpolypeptide comprising an enzymatic active domain of the invention asidentified herein is incubated together with an S. aureus suspensionhaving an initial OD₅₉₅ of 1±0.05, as assessed spectrophotometrically(Libra S22, Biochrom), in PBS buffer pH 7.4, 120 mM sodium chloride for30 min at 37° C. The drop in turbidity is calculated by subtracting theOD₅₉₅ after 30 min of incubation from the OD₅₉₅ before 30 min ofincubation. Within the context of the invention a polypeptide comprisingan enzymatic active domain of the invention as identified herein will besaid to have lytic activity if, when using this assay, a drop inturbidity of at least 10, 20, 30, 40, 50 or 60% is detected. Preferably,a drop in turbidity of at least 70% is detected. Preferably, apolypeptide comprising an enzymatic active domain of the inventionexhibits a lytic activity of at least 30, 40, 50, 60, 70, 80, 90, 100,150 or 200% or more of a lytic activity of S. aureus bacteriophage(I2638a endolysin (Ply2638 endolysin identified by SEQ ID NO: 2)preferably encoded by SEQ ID NO: 1. Preferably, the invention providesfor a composition comprising a first and a second compound, wherein saidfirst compound is a vasoconstrictive compound and said second compoundis a compound specifically targeting a bacterial cell, preferably a grampositive bacterial cell, wherein said second compound comprises one ormore enzymatic active domains exhibiting target bond specificity, andwherein said target bond is an essential bond in a peptidoglycan layerof said bacterial cell, preferably gram positive bacterial cell. Anessential bond in a peptidoglycan layer of a bacterial cell, preferablya gram-positive bacterial cell is defined herein as a linkage withinsaid peptidoglycan that is essential for said peptidoglycan to providesaid bacterial cell shape and a rigid structure resistance to osmoticshock. Preferably, said essential bond in a peptidoglycan layer of agram-positive bacterial cell is a bond between a D-alanine of the stempeptide and a glycine of the cross-bridge peptide (defined herein alsoas a bond between an N-terminal alanine and a glycine), a bond in apentaglycin cross-bridge (defined herein also as a pentaglycin bridgeglycyl-glycyl bond, a bond between an N-acetylmuramoyl and an L-alanineor a bond between an N-acetylmuramine and a N-acetylglucosamine orbetween a N-acetlyglucosamine and an N-acetylmuramine. Other preferredessential bonds in a peptidoglycan layer of a gram-positive bacterialcell are a bond in a gamma-glutamyl stem peptide, a bond between anL-alanyl-iso-D-glutamic acid in a stem peptide and a bond between aniso-D-glutamic acid-L-Lysine in a stem peptide.

Most native Staphylococcus bacteriophage endolysins exhibitingpeptidoglycan hydrolase activity consist of a C-terminal cellwall-binding domain (CBD), a central N-acetylmuramoyl-L-Alanine amidasedomain, and an N-terminal alanyl-glycyl endopeptidase domain withcysteine, histidine-dependent amidohydrolases/peptidase (CHAP) homology,or in case of Ply2638, of an N-terminal glycyl-glycine endopeptidasedomain with Peptidase_M23 homology, the latter three domains exhibitingpeptidoglycan hydrolase activity each with distinct target bondspecificity and generally named herein as enzymatically active domains.Preferably, said one or more enzymatic active domains is/are selectedfrom or is/are a permutation of a domain of the group consisting of acysteine, histidine dependent amidohydrolases/peptidase domain, anendopeptidase domain, an amidase domain and a glycosylhydrolase domain.Said glycosylhydrolase domain can be a muramidase domain or aglycosaminidase domain. Preferably, said CHAP domain cleaves a bondbetween an N-terminal alanyl and a glycyl within a peptidoglycan layer.More preferably, said CHAP domain specifically cleaves a bond between anN-terminal alanyl and a glycyl within a peptidoglycan layer. Preferably,said endopeptidase domain cleaves pentaglycin bridge glycyl-glycyl bondwithin a peptidoglycan layer. More preferably, said endopeptidase domainspecifically cleaves pentaglycin bridge glycyl-glycyl bond within apeptidoglycan layer. Preferably, said amidase domain cleaves a bondbetween a central N-acetlymuramoyl and an L-Alanine within apeptidoglycan layer. More preferably, said amidase domain specificallycleaves a bond between a central N-acetlymuramoyl and an L-Alaninewithin a peptidoglycan layer. Preferably, said murimidase domain cleavesa bond between an N-acetylmuramine and a N-acetylglucosamine within apeptidoglycan layer. More preferably, said murimidase domainspecifically cleaves a bond between an N-acetylmuramine and aN-acetylglucosamine within a peptidoglycan layer. Preferably, saidglucosaminidase domain cleaves a bond between an N-acetlyglucosamine andan N-acetylmuramine within a peptidoglycan layer. More preferably, saidglucosaminidase domain specifically cleaves a bond between anN-acetlyglucosamine and an N-acetylmuramine within a peptidoglycanlayer. Preferably said peptidoglycan layer is of a bacterial cell,preferably a gram positive bacterial cell, more preferably of aStaphylococcus, most preferably of a Staphylococcus Aureus. Preferably,the cleavage of a bond by an enzymatic active domain as defined hereinis specific if such a bond is hydrolyzed at least 2, 5, 10, 50 or a 100times more efficient with said enzymatic active domain as compared tothe hydrolyses of any other bond as defined herein above with saidenzymatic active domain.

Preferably, a CHAP domain encompassed within the invention originatesfrom Staphylococcus phage K, Staphylococcus phage Twort and/or S. aureusbacteriophage phi 11. Preferably, a CHAP domain encompassed within theinvention, is a domain that has at least 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQID NO: 10, 12 or 98 and/or is preferably encoded by SEQ ID NO: 9 or 11.Preferably, an endopeptidase domain encompassed within the inventionoriginates from S. aureus bacteriophage (2638a and/or S. simulans.Preferably, an endopeptidase domain encompassed by the invention has atleast 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99 or 100% identity with SEQ ID NO: 14 or 16 and/or ispreferably encoded by SEQ ID NO: 13 or 15. Preferably, an amidase domainencompassed within the invention originates from S. aureus bacteriophage(2638a or S. aureus bacteriophage phi 11. Preferably an amidase domainof the invention has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO:18 or 100 and/or is preferably encoded by SEQ ID NO: 17 or 99.

Preferably, the invention provides for a composition comprising a firstand a second compound, wherein said first compound is an avasoconstrictive compound and said second compound is a compoundspecifically targeting a bacterial cell, preferably a gram positivebacterial cell, wherein said second compound is a naturally occurring ormutant bacteriophage, a naturally occurring endolysin or a mutantpolypeptide.

A naturally occurring bacteriophage of the invention may be anybacteriophage specifically targeting and infecting a bacterial cell,preferably bacterial cell, preferably a Staphylococcus, most preferablya Staphylococcus aureus. Preferably, a naturally occurring bacteriophageof the invention is selected from, but not limited to, a groupconsisting of S. aureus bacteriophage Φ2638a, S. aureus bacteriophageΦ11, S. aureus bacteriophage ΦTwort, S. haemolyticus JCSC1435, S. aureusPhage K, S. warneri phage WMY, S. aureus phage NM3 and S. aureus80alpha. Said naturally occurring endolysin may be synthesized and/orpurified. A bacteriophage according to the invention may be a mutant,chimeric and/or recombinant bacteriophage. The person skilled in the artmay construct a bacteriophage of the invention by placing mutations inthe genome and/or deleting and/or inserting coding sequences or partsthereof into the genome using methods known in the art.

A naturally occurring endolysin may be any wild type or native endolysinexhibiting peptidoglycan hydrolase activity. Preferred is aStaphylococcus phage endolysin, preferably said Staphylococcus phageendolysin is selected from, but not limited to, the group consisting ofS. aureus bacteriophage Φ2638a endolysin, S. aureus bacteriophage Φ11endolysin, S. aureus bacteriophage ΦTwort endolysin, S. haemolyticusJCSC1435, S. aureus Phage K endolysin, S. warneri phage WMY endolysin,S. aureus phage NM3 endolysin and S. aureus 80alpha endolysin. Alsopreferred is S. simulans lysostaphin and/or a homologue of S. simulanslysostaphin such as S. capitis ALE-1 enzyme. Most native Staphylococcusbacteriophage endolysins exhibiting peptidoglycan hydrolase activityconsist of a C-terminal cell wall-binding domain (CBD), a centralN-acetylmuramoyl-L-Alanine amidase domain, and an N-terminalAlanyl-glycyl endopeptidase domain with CHAP homology, or in case ofPly2638, of an N-terminal endopeptidase domain with Peptidase_M23homology, the latter three domains exhibiting peptidoglycan hydrolaseactivity each with distinct target bond specificity and generally namedherein as enzymatically active domains.

A mutant polypeptide as encompassed within the invention may be achemically synthesized polypeptide or a recombinant or retrofittedpolypeptide produced in vitro. A retrofitted construct is defined hereinas a polynucleotide comprising heterologous nucleotide sequences. Asused herein the term heterologous sequence or heterologouspolynucleotide is one that is not naturally found operably linked asneighboring sequence of said first nucleotide sequence. As used herein,the term heterologous may mean recombinant. Recombinant refers to agenetic entity distinct from that generally found in nature. As appliedto a nucleotide sequence or nucleic acid molecule, this means that saidnucleotide sequence or nucleic acid molecule is the product of variouscombinations of cloning, restriction and/or ligation steps, and otherprocedures that result in the production of a construct that is distinctfrom a sequence or molecule found in nature. Preferably, a mutantpolypeptide to the invention comprises at least an enzymatic activedomain and a cell binding domain as defined herein.

An endolysin or mutant polypeptide of the invention may be in a purifiedform or may be comprised within a crude composition, preferably ofbiological origin, such as a bacterial lysate, yeast lysate, fungallysate, sonicate or fixate. Alternatively, said endolysin or mutantpolypeptide may be a chemically synthesized endolysin or polypeptide ora recombinant polypeptide produced in vitro.

An endolysin or mutant polypeptide of the invention preferably comprisesor consists of at least one enzymatic active domain and at least onecell binding domain and optionally a tag for ease of purification.Preferably, said tag is selected from, but is not limited to, the groupconsisting of a FLAG-tag, poly(His)-tag, HA-tag and Myc-tag. Morepreferably said tag is a 6×His-tag. Even more preferably, said tag is anN-terminal 6×His-tag (indicated herein as HXa) identical to SEQ ID NO:74 and preferably encoded by SEQ ID NO: 73).

Preferably, a cell wall-binding domain according to the invention islocated on the C-terminal side of the enzymatic active domain withinsaid naturally occurring endolysin or a mutant polypeptide. Preferably,said mutant naturally occurring or mutant polypeptide comprises at leasttwo or more enzymatic active domains with distinct target bondspecificities as distinct target bond specificities confer synergisticeffects. In an embodiment of the invention, a composition comprises atleast two distinct compounds targeting a bacterial cell, preferably agram positive bacterial cell, preferably a Staphylococcus, morepreferably a Staphylococcus aureus. Preferably said at least twodistinct compounds are naturally occurring endolysin, which areoptionally synthesized. Preferably said at least two distinct compoundsare recombinant polypeptides each comprising a distinct enzymatic activedomain and/or a different multiplicity of at least two distinctenzymatic active domains as defined herein below.

Preferably, the invention provides for a composition comprising a firstand a second compound, wherein said first compound is a vasoconstrictivecompound and said second compound is a compound specifically targeting abacterial cell, preferably a gram positive bacterial cell, wherein saidsecond compound is a recombinant polypeptide comprising a multiplicityof said one or more enzymatic active domains exhibiting target bondspecificity. “Multiplicity” is to be understood as a number of copiesand may be any integer varying from 1 to 20, preferably from 1 to 10,more preferably from 1 to 3, most preferably said multiplicity is 2,i.e. a duplicate. Polypeptides comprising a multiplicity of enzymaticactive domains show superior lytic activity as compared to polypeptidescomprising a single enzymatic active domain.

Preferably, said second compound is a polypeptide comprising and/orconsisting of an enzymatic active domain, a cell wall binding andoptionally a tag for ease of purification as defined herein, preferablysaid enzymatic active domain being a cysteine, histidine-dependentamidohydrolases/peptidase domain, an endopeptidase domain or an amidasedomain, and preferably polypeptide comprises a multiplicity of saidenzymatic active domain, preferably said multiplicity being 2, i.e. aduplicate. More preferably said polypeptide comprises and/or consists ofa duplicated amidase domain and a cell wall binding domain andoptionally a tag for ease of purification as defined herein, preferablysaid amidase is from S. aureus bacteriophage (I2638a endolysin and saidcell wall binding domain is of S. simulans lysostaphin. Most preferablysaid polypeptide comprises and/or consists of a duplicated endopeptidasedomain and a cell wall binding domain and optionally a tag for ease ofpurification as defined herein, preferably said endopeptidase domain isa Peptidase_M23 domain of S. simulans lysostaphin and said cell wallbinding domain is of S. simulans lysostaphin.

Preferably, said second compound is a polypeptide has at least 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99or 100% identity with SEQ ID NO: SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16,18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52,54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 76, 78, 80, 82, 84, 86, 88, 90,92, 94, 98, 100 and 101 and/or is encoded by a polynucleotide having atleast 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99 or 100% identity to any of SEQ ID NO: 1, 3, 5, 7, 9, 11,13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47,49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 75, 77, 79, 81, 83, 85,87, 89, 91, 93, 97 or 99. Preferably, said polypeptide has at least 80,81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,99 or 100% identity with SEQ ID NO: 28, 34, 46, 52, 58, 70, 84 or 101;more preferably, said polypeptide has at least 80, 81, 82, 83, 84, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identitywith SEQ ID NO: 28, 46, 52, 70, 84 or 101; even more preferably, saidpolypeptide has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO: 46, 70,84 or 101; most preferably said polypeptide has at least 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%identity with SEQ ID NO: 70 or 101.

In all embodiments of the invention, the second compound according tothe invention may be an endolysin polypeptide that comprises a proteintransduction domain enabling the endolysin polypeptide to enter a cellthat harbors an intracellular bacterium. Protein transduction domainsare extensively described in EP158880.3 and these are preferred proteintransduction domains. When the second compound according to theinvention is an endolysin polypeptide comprising a protein transductiondomain enabling the endolysin polypeptide to enter a cell that harborsan intracellular bacterium, treatment preferably comprisesadministration of an effective amount of an agent that increases theintracellular pH of a host cell and/or of an intracellular compartmentof a host cell, wherein the increase in pH activates a non-replicatingintracellular bacterium and the endolysin kills the activatedintracellular bacterium. Such treatment is extensively described inEP15158880.3, which is herein incorporated by reference.

All embodiments of the invention relating to preventing, delaying and/orcuring of telangiectasia, erythema and/or flushing, preferablytelangiectasia, erythema and/or flushing associated with inflammationinduced vasodilatation, can be combined with the combination treatmentfor topical dermatitis as described in WO2015/005787, which is hereinincorporated by reference. WO2015/005787 describes preventing, delayingand/or curing of atopic dermatitis, preferably eczema using acomposition comprising a first and a second compound, wherein said firstcompound is an anti-inflammatory compound and said second compound is acompound specifically targeting a bacterial cell, preferably a grampositive bacterial cell. The second compound may be a second compoundaccording to the invention.

Preferably, the invention provides for a composition comprising a firstand a second compound, wherein said first compound is a vasoconstrictivecompound and said second compound is a compound specifically targeting abacterial cell, preferably a gram positive bacterial cell, wherein saidfirst compound is a sympathomimetic compound. A sympathomimetic compound(also referred to as sympathomimetic drug) is a stimulant compound whichmimics the effects of an agonist of the sympathetic nervous system suchas a catecholamine. (epinephrine (adrenaline), norepinephrine(noradrenaline), dopamine, etc.). The mechanism of a sympathomimeticdrug can be direct-acting, such as an α-adrenergic agonist, β-adrenergicagonist, and a dopaminergic agonist; or indirect-acting, such as an MAOI(MonoAmineOxidase Inhibitor), a COMT (Catechol-O-Methyl Transferase)inhibitor, a release stimulant, and a reuptake inhibitor that increasesthe level of an endogenous catecholamine.

The first compound preferably is a sympathomimetic compound selectedfrom the group consisting of alpha(1)- and alpha(2)-adrenergicreceptor-mediated vasoconstrictive compounds, such as Brimonidine,Tetrahydrozoline and Oxymetazoline; 25I-NBOMe; Amphetamines;5-methoxy-α-methyltryptamine 5-MeO-AMT (5-methoxy-α-methyltryptamine);Antihistamines; Caffeine; Cocaine; DOM(2,5-dimethoxy-4-methylamphetamine); LSA (Lysergic acid amide);Methylphenidate; Mephedrone; Phenylephrine; Propylhexedrine;Pseudoephedrine; Psycho stimulants; Benzylpiperazine; Cathine; Cathinon;Ephedrine; Lisdexamfetatime; maprotiline; Methamphetamine;methcathinone; methylenedioxypyrovalerone; methylphenidate (Ritalin);4-methylaminorex; Pemoline; Phenmetrazine; and Propylhexedrine; or acombination of two or more of these. A more preferred first compound isa vasoconstrictive compound selected from the group consisting ofalpha(1)- and alpha(2)-adrenergic agonists such as Brimonidine,Tetrahydrozoline and Oxymetazoline. A most preferred first compound is avasoconstrictive compound selected from the group consisting ofBrimonidine, Tetrahydrozoline and Oxymetazoline. The compounds listedhere above are herein referred to as a vasoconstrictive compoundaccording to the invention or a sympathomimetic compound according tothe invention and are applicable to all embodiments of the invention.

In all embodiments of the invention, the second compound specificallytargeting a bacterial cell, preferably a gram positive bacterial cellmay be comprised of a combination of a source of a first enzymaticactive domain and a source of a second enzymatic active domain, whereinsaid first and second enzymatic active domains exhibit distinct targetbond specificities and are comprised on a distinct first and secondpolypeptide, i.e. said first enzymatic active domain is comprised on afirst polypeptide and said second enzymatic domain is comprised on asecond polypeptide, wherein said first and second polypeptide each havea distinct amino acid sequence. In addition, the second compoundaccording to the invention may be comprised of a combination of a sourceof a first enzymatic active domain, a source of a second enzymaticactive domain and a source of a third enzymatic active domain, whereinsaid first, second and third enzymatic active domain exhibit distincttarget bond specificities and are comprised on a distinct first, secondand third polypeptide, i.e. said first enzymatic active domain iscomprised on a first polypeptide, said second enzymatic domain iscomprised on a second polypeptide, and said third enzymatic domain iscomprised on a third polypeptide, wherein said first, second and thirdpolypeptide each have a distinct amino acid sequence. Furthermore, thesecond compound according to the invention may be comprised of acombination of a source of a first enzymatic active domain, a source ofa second enzymatic active domain, a source of a third enzymatic activedomain, and a source of a further enzymatic active domain, wherein saidfirst, second, third and further enzymatic active domain exhibitdistinct target bond specificities and are comprised on a distinctfirst, second, third and further polypeptide, i.e. said first enzymaticactive domain is comprised on a first polypeptide, said second enzymaticdomain is comprised on a second polypeptide, said third enzymatic domainis comprised on a third polypeptide, and said further enzymatic activedomain is comprised on a further polypeptide, wherein said first,second, third and further polypeptide each have a distinct amino acidsequence. A further enzymatic active domain is meant herein as a fourth,fifth, sixth, seventh, eighth, ninth, tenth or more enzymatic activedomain, preferably a fourth enzymatic active domain. A furtherpolypeptide is meant herein as a fourth, fifth, sixth, seventh, eighth,ninth, tenth or more polypeptide, preferably a fourth polypeptide.

The inventors surprisingly found for the second compound according tothe invention, that simultaneous application of two or moreenzymatically active domains with distinct target bond specificitiesconfers synergistic effects. Surprisingly, this works not only whenenzymatically active domains with different specificities are located onthe same molecule as in native Staphylococcus endolysins, but works alsowhen the enzymatically active domains with different specificities areseparated on distinct polypeptides. The benefit of having distinctenzymatic active domains located on separate individual polypeptides isthat the resulting polypeptides are smaller which can be more easilyproduced. Furthermore, these smaller polypeptides have better diffusionproperties in specific environments and can be more resistant todegradation and feature higher thermostability. Another advantage isthat independent distinct enzymatic active domains located on separatedistinct polypeptide molecules can be mixed and pooled in variablecompositions, at a ratio that is best suited to hydrolyse the specificbacterial target cells. The second compound according to the inventioncomprised of a combination as described herein can be supplementedand/or complemented by the use of virtually any functional enzymaticactive domain with virtually any target bond specificity from manydifferent origins including phage lysins, bacteriocins, autolysins, orany other cell wall lytic enzymes.

Within the context of the second compound according to the invention ‘acombination’ means that a source of a first enzymatic active domain anda source of a second enzymatic active domain are contemplated andencompassed. In addition, within the context of the second compoundaccording to invention ‘a combination’ means that a source of a firstenzymatic active domain, a source of a second enzymatic active domainand optionally a source of a third and/or further enzymatic activedomain are contemplated and encompassed. Each source may be together orpresent together or combined together or physically in contact with theother source forming one single composition. Each source mayalternatively be comprised within a distinct composition. However theinvention provides the insight that both sources of a first and a secondenzymatic active domain are preferably needed or are used in order toget an effect of the invention as defined herein.

If each source is not present in a same single composition, each sourceand/or each distinct composition comprising a source of a combinationencompassing the second compound according to the invention may be usedsequentially or simultaneously. ‘A source of a first enzymatic activedomain’, ‘a source of a second enzymatic active domain’, ‘a source of athird enzymatic active domain’ and ‘a source of a further enzymaticactive domain’ preferably comprises a protein-based source, i.e. apolypeptide, a protein, digest of a protein and/or fragment of a proteinor digest, or a source not being protein based, i.e. a nucleic acidencoding a protein or derived peptide or protein fragment. Below wedefine preferred sources of a first enzymatic active domain, a source ofa second enzymatic active domain, a source of a third enzymatic activedomain and a source of a further enzymatic active domain that areencompassed by the invention. When the second compound according to theinvention relates to a combination of a source of a first enzymaticactive domain, a source of a second enzymatic active domain andoptionally a source of a third and/or further enzymatic active domain,each of the sources of a first enzymatic active domain defined hereinmay be combined with each of the sources of a second and optionallythird and/or further enzymatic active domain defined herein. It is alsoencompassed by the invention to use a combination of a source of a firstenzymatic active domain being protein-based with a source of a secondand optionally a third and/or further enzymatic active domain being notprotein-based, and vice versa.

‘Comprised on distinct polypeptides’ is meant herein as any of saidfirst, second and optionally third and/or further enzymatic activedomain is comprised on a polypeptide which is distinct from thepolypeptide that any of the other of said first, second and optionallythird and/or further enzymatic active domain is comprised on.

In all embodiments according to the invention, a polypeptide can be anatural polypeptide or an isolated polypeptide, preferably an isolatedpolypeptide. A nucleic acid according to the invention may be a naturalnucleic acid or an isolated nucleic acid, preferably an isolated nucleicacid. A nucleic acid construct according to the invention can be anatural or an isolated construct, preferably an isolated nucleic acidconstruct.

Preferably, a first, a second and optionally a third and/or furtherenzymatic active domain together encompassing the second compoundaccording to the invention is a domain selected from the groupconsisting of a cysteine, histidine-dependent amidohydrolases/peptidase(CHAP) domain, an endopeptidase domain, and an amidase domain; allpreferably as described previously herein.

Preferably, a first, second, third and/or further polypeptide togetherencompassing the second compound according to the invention comprises adifferent multiplicity of a first, second, third and/or furtherenzymatic active domain according to the invention. A “multiplicity” isherein defined as a number of copies. A “different multiplicity” isdefined herein as a multiplicity or number of copies of a specificenzymatic active domain according to the invention, i.e. a first,second, third or further enzymatic active domain as defined herein,comprised within a specific polypeptide of the invention, i.e. a first,second, third or further polypeptide as defined herein, to be differentform a multiplicity or number of copies of that same enzymatic activedomain within another polypeptide of the combination encompassing thesecond compound of the invention. For example, a combinationencompassing the second compound of the invention comprises a firstpolypeptide comprising a specific number of copies of a first enzymaticactive domain, and a second polypeptide comprising a different number ofcopies of said first enzymatic active domain. Furthermore, said firstpolypeptide of said exemplified combination encompassing the secondcompound of the invention may further comprise a specific number ofcopies of second enzymatic active domain, which is different from thenumber of copies of said second enzymatic active domain as comprised onsaid second polypeptide of said combination. Furthermore, any furtherpolypeptide of said exemplified combination encompassing the secondcompound of the invention may comprise a number of copies of furtherenzymatic active domain, which is different from the number of copies ofsaid further enzymatic active domain as comprised on said first andsecond polypeptide of said combination. Although a combination ofdistinct polypeptides each comprising a single distinct enzymatic activedomain showed synergistic lytic activity as compared to the lyticactivity of each separate polypeptide, it was surprisingly found by thepresent inventors that polypeptides comprising a multiplicity ofenzymatic active domains show superior lytic activity as compared topolypeptides comprising a single enzymatic active domain.

Moreover, a combination of distinct enzymatic domains on distinctpolypeptides wherein at least one of said distinct polypeptidescomprises a multiplicity of enzymatic active domains was found superiorover a combination wherein all said distinct polypeptides comprise asingle distinct enzymatic active domain. Moreover, a combinationencompassing the second compound according to the invention, wherein afirst, second, third and/or further polypeptide comprise a multiplicityof a first, second, third and/or further enzymatic active domainaccording to the invention, respectively, was found superior over acombination encompassing the second compound according to the invention,wherein said first, second, third and/or further polypeptide comprise asingle copy of said first, second, third and/or further enzymatic activedomain, respectively, and preferably wherein said multiplicity, asdefined herein, is 2, i.e. a duplicate. In a preferred embodiment, thesynergistic effect of a combination encompassing the second compoundaccording to the invention, wherein a first, second, third and/orfurther polypeptide according to the invention comprise a multiplicityof a first, second, third and/or further enzymatic active domainaccording to the invention, respectively, was found superior over acombination encompassing the second compound according to the invention,wherein said first, second, third and further polypeptide comprise asingle copy of said first, second, third and further enzymatic activedomain, respectively, and preferably wherein said multiplicity, asdefined herein below, is 2, i.e. a duplicate.

Preferably, a first and/or second polypeptide of a combinationencompassing the second compound according to the invention, comprises adifferent multiplicity of a first and/or second enzymatic active domainaccording to the invention. Multiplicity of said first and second domainis defined as previously herein as a number of copies, preferablyindicated by k, l, n and p, of said first and second domain indicated asfollows:

-   k indicates the number of copies of said first enzymatic active    domain on said first polypeptide;-   l indicates the number of copies of said second enzymatic active    domain on said first polypeptide;-   n indicates the number of copies of said first enzymatic active    domain on said second polypeptide;-   p indicates the number of copies of said second enzymatic active    domain on said second polypeptide;    and wherein k and p are independent integers from 1-10, 1-9, 1-8,    1-7, 1-6, 1-5, 1-4, 1-3, or preferably 1-2, and l and n are    independent integers from 0-10, 0-9, 0-8, 0-7, 0-6, 0-5, 0-4, 0-3,    or preferably 0-2, and wherein k is a different integer than n    and/or l is a different integer than p, most preferably k and p are    2 and l and n are 0.

Preferably, a first, second and third polypeptide encompassing thesecond compound of the invention comprise a different multiplicity of afirst, second and third enzymatic active domain according to theinvention.

Multiplicity of said first, second and third domain is defined aspreviously herein as a number of copies, preferably indicated by k, l,m, n, p, q, r, s and t, of said first, second and third domain indicatedas follows:

-   k indicates the number of copies of said first enzymatic active    domain on said first polypeptide;-   l indicates the number of copies of said second enzymatic active    domain on said first polypeptide;-   m indicates the number of copies of said third enzymatic active    domain on said first polypeptide;-   n indicates the number of copies of said first enzymatic active    domain on said second polypeptide;-   p indicates the number of copies of said second enzymatic active    domain on said second polypeptide;-   q indicates the number of copies of said third enzymatic active    domain on said second polypeptide;-   r indicates the number of copies of said first enzymatic active    domain on said third polypeptide;-   s indicates the number of copies of said second enzymatic active    domain on said third polypeptide;-   t indicates the number of copies of said third enzymatic active    domain on said third polypeptide;    and wherein k, p and t are independent integers from 1-10, 1-9, 1-8,    1-7, 1-6, 1-5, 1-4, 1-3, or preferably 1-2, and l, m, n, q, r, and s    are independent integers from 0-10, 0-9, 0-8, 0-7, 0-6, 0-5, 0-4,    0-3, or preferably 0-2, and wherein k is a different integer than n    and/or r, and/or l is a different integer than p and/or s, and/or t    is a different integer than m or q, most preferably k, p and t are 2    and l, m, n, q, r, and s are 0.

Preferably, a first, second, third and further polypeptide encompassingthe second compound of the invention comprise a different multiplicityof a first, second, third and further enzymatic active domain accordingto the invention. Multiplicity of said further enzymatic active domainin view of said first, second and third enzymatic active domain is to beconstrued herein in an analogous manner as defined herein above for afirst, second and third enzymatic active domain.

Preferably a first, second, third or further polypeptide encompassingthe second compound according to the invention has a length of at least140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270,280, 290, 300, 310, 320 or 330 amino acids and/or a length of at most850, 800, 750, 700, 650, 600, 550, 500, 490, 480, 470, 460, 450, 440,430, 420, 410, 400, 390, 380 or 370 amino acids. More preferably, afirst, second or third polypeptide encompassing the second compoundaccording to the invention has a length of 140-850, 140-800, 140-750,140-700, 140-650, 140-600, 140-550 140-500, 140-490, 140-480, 140-470,140-460, 140-450, 140-440, 140-430, 140-420, 140-410, 140-400, 140-390,140-380, 140-370, 150-850, 160-850, 170-850, 180-850, 190-850, 200-850,210-850, 220-850, 230-850, 240-850, 250-850, 260-850, 270-850, 280-850,290-850, 300-850, 310-850, 320-850 or 330-850 amino acids.

Preferably a first and second polypeptide encompassing the secondcompound according to the invention each have a length of at least 140,150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280,290, 300, 310, 320 or 330 amino acids and/or a length of at most 800,850, 700, 650, 600, 550, 500, 490, 480, 470, 460, 450, 440, 430, 420,410, 400, 390, 380 or 370 amino acids. More preferably, a first andsecond polypeptide according to the invention each have a length of140-850, 140-800, 140-750, 140-700, 140-650, 140-600, 140-550 140-500,140-490, 140-480, 140-470, 140-460, 140-450, 140-440, 140-430, 140-420,140-410, 140-400, 140-390, 140-380, 140-370, 150-850, 160-850, 170-850,180-850, 190-850, 200-850, 210-850, 220-850, 230-850, 240-850, 250-850,260-850, 270-850, 280-850, 290-850, 300-850, 310-850, 320-850 or 330-850amino acids.

Preferably a first, second and third polypeptide encompassing the secondcompound according to the invention each have a length of at least 140,150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280,290, 300, 310, 320 or 330 amino acids and/or a length of at most 800,850, 700, 650, 600, 550, 500, 490, 480, 470, 460, 450, 440, 430, 420,410, 400, 390, 380 or 370 amino acids. More preferably, a first, secondand third polypeptides encompassing the second compound according to theinvention each have a length of 140-850, 140-800, 140-750, 140-700,140-650, 140-600, 140-550 140-500, 140-490, 140-480, 140-470, 140-460,140-450, 140-440, 140-430, 140-420, 140-410, 140-400, 140-390, 140-380,140-370, 150-850, 160-850, 170-850, 180-850, 190-850, 200-850, 210-850,220-850, 230-850, 240-850, 250-850, 260-850, 270-850, 280-850, 290-850,300-850, 310-850, 320-850 or 330-850 amino acids.

Preferably a first, second, third and further polypeptide encompassingthe second compound according to the invention each have a length of atleast 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260,270, 280, 290, 300, 310, 320 or 330 amino acids and/or a length of atmost 800, 850, 700, 650, 600, 550, 500, 490, 480, 470, 460, 450, 440,430, 420, 410, 400, 390, 380 or 370 amino acids. More preferably, afirst, second, third and further polypeptides encompassing the secondcompound according to the invention each have a length of 140-850,140-800, 140-750, 140-700, 140-650, 140-600, 140-550 140-500, 140-490,140-480, 140-470, 140-460, 140-450, 140-440, 140-430, 140-420, 140-410,140-400, 140-390, 140-380, 140-370, 150-850, 160-850, 170-850, 180-850,190-850, 200-850, 210-850, 220-850, 230-850, 240-850, 250-850, 260-850,270-850, 280-850, 290-850, 300-850, 310-850, 320-850 or 330-850 aminoacids.

An embodiment provides a combination of a source of a first and a secondenzymatic active domain encompassing the second compound according tothe invention, wherein said first and second enzymatic active domainsare comprised on distinct, first and second polypeptides of theinvention, wherein said first polypeptide is free of said secondenzymatic active domain and said second polypeptide is free of saidfirst enzymatic active domain. Moreover, provided is a combinationaccording to the invention, wherein l and n are 0.

Another embodiment provides a combination of a source of a first, secondand third enzymatic active domain encompassing the second compoundaccording to the invention, wherein said first, second and thirdenzymatic active domains are comprised on distinct, first, second andthird polypeptides, wherein said first polypeptide is free of saidsecond and third enzymatic active domain, said second polypeptide isfree of said first and third enzymatic active domain, and said thirdpolypeptide is free of said first and second enzymatic active domain.Moreover, provided is a combination according to the invention, whereinl, m, n, q, r and s are 0. Even more preferably, the invention providesa combination encompassing the second compound according to theinvention, wherein l, m, n, q, r and s are 0 and k, p and t are 2.

Another embodiment provides a combination of a source of a first,second, third and further enzymatic active domain encompassing thesecond compound according to the invention, wherein said first, second,third and further enzymatic active domains are comprised on a distinct,first, second, third and further polypeptide, respectively, whereinpreferably said first polypeptide is free of said second, third andfurther enzymatic active domain;

-   -   preferably said second polypeptide is free of said first, third        and further enzymatic active domain;    -   preferably said third polypeptide is free of said first, second        and further enzymatic active domain; and,    -   preferably said further polypeptide is free of said first,        second and third enzymatic active domain.

Preferably said first, second, third and further enzymatic active domainare comprised within said first, second, third and further polypeptide,respectively, in duplicate, i.e. wherein the multiplicity as identifiedherein is 2. Also encompassed is a combination encompassing the secondcompound according to the invention, wherein a first, second and/orthird polypeptide according to the invention are not free of a first,second and/or third enzymatic active domain according to the invention,but said first, second and/or third polypeptide differ in multiplicityof said first, second and/or third enzymatic active domain. Moreover,encompassed is a combination encompassing the second compound accordingto the invention, wherein at least one of k, l, m, n p, q, r, s or t is2 and wherein any of the other k, 1, m, n p, q, r, s and/or t is 1 or 0.Preferred is a combination encompassing the second compound according tothe invention, wherein a first, second, third and/or further polypeptideis a polypeptide that has at least 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with apolypeptide selected from the group consisting of SEQ ID NO: 26, 28, 30,32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66,68, 70, 72, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 98 or 100.

Within the context of the invention, several preferred, non-limiting,combinations encompassing the second compound according to the inventionare envisaged, which are listed here below.

Preferred is a combination of a source of first enzymatic active domainand a second enzymatic active domain, wherein said first and secondenzymatic active domains are comprised on distinct first and secondpolypeptides, and wherein said first enzymatic active domain is acysteine, histidine-dependent amidohydrolases/peptidase domain and saidsecond enzymatic active domain is an endopeptidase domain or whereinsaid first enzymatic active domain is a cysteine, histidine-dependentamidohydrolases/peptidase domain and said second enzymatic active domainis amidase domain or wherein said first enzymatic active domain is anendopeptidase domain and said second enzymatic active domain is amidasedomain, wherein said distinct first and second each further comprises acell wall-binding domain, and wherein each of said distinct first andsecond polypeptides comprises a multiplicity of said first or secondenzymatic active domain, preferably said multiplicity being 2, i.e. aduplicate.

Also preferred is a combination of a source of first and secondenzymatic active domain, wherein said first and second enzymatic activedomains are comprised on distinct first and second polypeptides, andwherein said first enzymatic domain is histidine-dependentamidohydrolases/peptidase domain and said second enzymatic active domainis an endopeptidase domain or said first enzymatic active domain is acysteine, histidine-dependent amidohydrolases/peptidase domain and saidsecond enzymatic active domain is amidase domain or said first enzymaticactive domain is an endopeptidase domain and said second enzymaticactive domain is amidase domain, and wherein said first and secondpolypeptide each further comprise a cell wall binding domain.

Also preferred is a combination of a source of first enzymatic activedomain and a second enzymatic active domain, wherein said first andsecond enzymatic active domains are comprised on distinct first andsecond polypeptides, and wherein said first enzymatic active domain is acysteine, histidine-dependent amidohydrolases/peptidase domain and saidsecond enzymatic active domain is an endopeptidase domain, and whereinsaid combination further comprises a source of a third enzymatic activedomain comprised on a distinct third polypeptide, wherein said thirdenzymatic active domain is an amidase domain and said distinct first,second and third polypeptide each further comprises a cell wall-bindingdomain, and wherein each of said distinct first, second and thirdpolypeptides comprises a multiplicity of said first, second or thirdenzymatic active domain, preferably said multiplicity being 2, i.e. aduplicate.

Also preferred is a combination of a source of first, second and thirdenzymatic active domain, wherein said first, second and third enzymaticactive domains are comprised on distinct first, second and thirdpolypeptides, and wherein said first enzymatic domain ishistidine-dependent amidohydrolases/peptidase domain, said secondenzymatic active domain is an endopeptidase domain and said thirdenzymatic active domain is an amidase domain, and wherein said first,second and third polypeptide each further comprise a cell wall bindingdomain.

Also preferred is a combination wherein, a first enzymatic active domainaccording to the invention has at least 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQID NO: 10 and a second enzymatic active domain according to theinvention as at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO: 16.

Also preferred is a combination wherein, a first enzymatic active domainaccording to the invention has at least 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQID NO: 10 and a second enzymatic active domain according to theinvention as at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO: 18.

Also preferred is a combination wherein, a first enzymatic active domainaccording to the invention has at least 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQID NO: 16 and a second enzymatic active domain according to theinvention as at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO: 18.

Also preferred is a combination wherein, a first polypeptide accordingto the invention has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO:34 and a second polypeptide according to the invention as at least 80,81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,99 or 100% identity with SEQ ID NO: 46.

Also preferred is a combination wherein, a first polypeptide accordingto the invention has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO:34 and a second polypeptide according to the invention as at least 80,81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,99 or 100% identity with SEQ ID NO: 28.

Also preferred is a combination wherein, a first polypeptide accordingto the invention has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO:46 and a second polypeptide according to the invention as at least 80,81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,99 or 100% identity with SEQ ID NO: 28.

Also preferred is a combination wherein, a first polypeptide accordingto the invention has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO:58 and a second polypeptide according to the invention as at least 80,81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,99 or 100% identity with SEQ ID NO: 70.

Also preferred is a combination wherein, a first polypeptide accordingto the invention has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO:58 and a second polypeptide according to the invention as at least 80,81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,99 or 100% identity with SEQ ID NO: 52.

More preferred is a combination wherein, a first polypeptide accordingto the invention has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO:70 and a second polypeptide according to the invention as at least 80,81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,99 or 100% identity with SEQ ID NO: 52.

Also preferred is a combination wherein, a first polypeptide accordingto the invention has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO:58 and a second polypeptide according to the invention as at least 80,81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,99 or 100% identity with SEQ ID NO: 46.

Also preferred is a combination wherein, a first polypeptide accordingto the invention has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO:58 and a second polypeptide according to the invention as at least 80,81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,99 or 100% identity with SEQ ID NO: 28.

Also preferred is a combination wherein, a first polypeptide accordingto the invention has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO:70 and a second polypeptide according to the invention as at least 80,81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,99 or 100% identity with SEQ ID NO: 34.

More preferred is a combination wherein, a first polypeptide accordingto the invention has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO:70 and a second polypeptide according to the invention as at least 80,81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,99 or 100% identity with SEQ ID NO: 28.

Also preferred is a combination, wherein a first polypeptide accordingto the invention has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO:52 and a second polypeptide according to the invention as at least 80,81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,99 or 100% identity with SEQ ID NO: 34.

Also preferred is a combination wherein, a first polypeptide accordingto the invention has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO:52 and a second polypeptide according to the invention as at least 80,81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,99 or 100% identity with SEQ ID NO: 46.

Also preferred is a combination wherein, a first enzymatic active domainaccording to the invention has at least 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQID NO: 10, a second enzymatic active domain according to the inventionas at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99 or 100% identity with SEQ ID NO: 16 and a thirdenzymatic active domain according to the invention has at least 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99or 100% identity with SEQ ID NO: 18.

Also preferred is a combination wherein, a first polypeptide accordingto the invention has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO:34, a second polypeptide according to the invention as at least 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99or 100% identity with SEQ ID NO: 46 and a third polypeptide according tothe invention has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO: 28.

Also preferred is a combination wherein, a first polypeptide accordingto the invention has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO:32, a second polypeptide according to the invention as at least 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99or 100% identity with SEQ ID NO: 44 and a third polypeptide according tothe invention has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO: 26.

Also preferred is a combination wherein, a first polypeptide accordingto the invention has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO:34, a second polypeptide according to the invention as at least 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99or 100% identity with SEQ ID NO: 46 and a third polypeptide according tothe invention has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO: 26.

Also preferred is a combination wherein, a first polypeptide accordingto the invention has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO:36, a second polypeptide according to the invention as at least 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99or 100% identity with SEQ ID NO: 48 and a third polypeptide according tothe invention has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO: 30.

Also preferred is a combination wherein, a first polypeptide accordingto the invention has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO:32, a second polypeptide according to the invention as at least 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99or 100% identity with SEQ ID NO: 46 and a third polypeptide according tothe invention has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO: 26.

Also preferred is a combination wherein, a first polypeptide accordingto the invention has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO:34, a second polypeptide according to the invention as at least 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99or 100% identity with SEQ ID NO: 44 and a third polypeptide according tothe invention has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO: 26.

Also preferred is a combination wherein, a first polypeptide accordingto the invention has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO:32, a second polypeptide according to the invention as at least 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99or 100% identity with SEQ ID NO: 44 and a third polypeptide according tothe invention has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO: 28.

Also preferred is a combination wherein, a first polypeptide accordingto the invention has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO:32, a second polypeptide according to the invention as at least 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99or 100% identity with SEQ ID NO: 46 and a third polypeptide according tothe invention has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO: 28.

Also preferred is a combination wherein, a first polypeptide accordingto the invention has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO:34, a second polypeptide according to the invention as at least 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99or 100% identity with SEQ ID NO: 44 and a third polypeptide according tothe invention has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO: 28.

Also preferred is a combination wherein, a first polypeptide accordingto the invention has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO:58, a second polypeptide according to the invention as at least 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99or 100% identity with SEQ ID NO: 70 and a third polypeptide according tothe invention has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO: 52.

Also preferred is a combination wherein, a first polypeptide accordingto the invention has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO:58, a second polypeptide according to the invention as at least 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99or 100% identity with SEQ ID NO: 70 and a third polypeptide according tothe invention has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO: 50.

Also preferred is a combination wherein, a first polypeptide accordingto the invention has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO:56, a second polypeptide according to the invention as at least 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99or 100% identity with SEQ ID NO: 68 and a third polypeptide according tothe invention has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO: 50.

Also preferred is a combination wherein, a first polypeptide accordingto the invention has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO:60, a second polypeptide according to the invention as at least 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99or 100% identity with SEQ ID NO: 72 and a third polypeptide according tothe invention has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO: 54.

Also preferred is a combination wherein, a first polypeptide accordingto the invention has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO:56, a second polypeptide according to the invention as at least 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99or 100% identity with SEQ ID NO: 70 and a third polypeptide according tothe invention has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO: 50.

Also preferred is a combination wherein, a first polypeptide accordingto the invention has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO:58, a second polypeptide according to the invention as at least 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99or 100% identity with SEQ ID NO: 68 and a third polypeptide according tothe invention has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO: 50.

Also preferred is a combination wherein, a first polypeptide accordingto the invention has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO:56, a second polypeptide according to the invention as at least 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99or 100% identity with SEQ ID NO: 68 and a third polypeptide according tothe invention has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO: 52.

Also preferred is a combination wherein, a first polypeptide accordingto the invention has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO:65, a second polypeptide according to the invention as at least 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99or 100% identity with SEQ ID NO: 70 and a third polypeptide according tothe invention has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO: 52.

Also preferred is a combination wherein, a first polypeptide accordingto the invention has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO:58, a second polypeptide according to the invention as at least 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99or 100% identity with SEQ ID NO: 68 and a third polypeptide according tothe invention has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO: 52.

Also preferred is a combination wherein, a first polypeptide accordingto the invention has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO:58, a second polypeptide according to the invention as at least 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99or 100% identity with SEQ ID NO: 70 and a third polypeptide according tothe invention has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO: 28.

Also preferred is a combination wherein, a first polypeptide accordingto the invention has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO:34, a second polypeptide according to the invention as at least 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99or 100% identity with SEQ ID NO: 70 and a third polypeptide according tothe invention has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO: 52.

Also preferred is a combination wherein, a first polypeptide accordingto the invention has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO:58, a second polypeptide according to the invention as at least 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99or 100% identity with SEQ ID NO: 46 and a third polypeptide according tothe invention has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO: 52.

Also preferred is a combination wherein, a first polypeptide accordingto the invention has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO:34, a second polypeptide according to the invention as at least 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99or 100% identity with SEQ ID NO: 46 and a third polypeptide according tothe invention has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO: 52.

Also preferred is a combination wherein, a first polypeptide accordingto the invention has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO:58, a second polypeptide according to the invention as at least 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99or 100% identity with SEQ ID NO: 46 and a third polypeptide according tothe invention has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO: 28.

Also preferred is a combination wherein, a first polypeptide accordingto the invention has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO:34, a second polypeptide according to the invention as at least 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99or 100% identity with SEQ ID NO: 70 and a third polypeptide according tothe invention has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO: 28.

It is to be understood that a combination as described hereinencompassing the second compound according to the invention includesmixtures of a source of a first, a source of a second and optionally asource of a third and/or further enzymatic active domain according to invarying ratios. Preferably, such combination comprises a source a firstand a source a second enzymatic active domain according to theinvention, wherein said first and second enzymatic active domain arepresent in equimolar amounts. Also preferred is a combination comprisinga source a first, a source a second and a source a third enzymaticactive domain according to the invention, wherein said first, second andthird enzymatic active domain are present in equimolar amounts. Alsopreferred is a combination comprising a source of a first, a source of asecond, a source of a third and a source of a further enzymatic activedomain according to the invention, wherein said first, second, third andfurther enzymatic active domain are present in equimolar amounts.

In a second aspect, the invention provides for a kit of partscomprising:

-   -   a) a first vial containing a first composition comprising a        first compound as defined in the first aspect of the invention;        and,    -   b) a second vial containing a second composition comprising a        second compound as defined in the first aspect of the invention;        and optionally,    -   c) instructions for use, preferably comprising a dosage regime.

Preferably, said kit of parts, more specifically said first and secondcomposition of said kit of parts, is for use as a medicament, preferablyfor use in treatment of rosacea, more preferably for use in thetreatment of telangiectasia, erythema and/or flushing, preferablytelangiectasia, erythema and/or flushing associated with inflammationinduced vasodilatation, as further detailed herein. A dosage regime isto be understood herein as an instruction for administration to anindividual in the need thereof, preferably an instruction indicating anadministration route, administration frequency and administrationdosage, and optionally an instruction for admixing said first and secondcompound just before administration, as required for treatment,preferably required for treatment of rosacea, more preferably for use inthe treatment of telangiectasia, erythema and/or flushing, preferablytelangiectasia, erythema and/or flushing associated with inflammationinduced vasodilatation, as further detailed herein. Preferredadministration routes, frequencies and dosages are further detailedherein. In an embodiment, said first composition according to a secondaspect and/or said second composition according to a second aspect ofthe invention is administered separately, preferably as part of anoverall treatment regimen. In an alternative embodiment, said firstcomposition according to a second aspect and said second compositionaccording to a second aspect of the invention are stored separately, andadmixed just before administration. Preferably, “just before” is to beunderstood herein as less than 120, 60, 30, 15, 5, 4, 3, 2 or 1 minutesbefore administration, preferably less than 5 minutes beforeadministration.

Said first and said second vial may be any vial, bottle, tube, ampoule,container, flask or the like, suitable for storing said first and secondcomposition as defined herein, respectively. Preferably said firstand/or second vial has a volume of between 0.1 and 500 mL, preferablybetween 1 and 100 mL, more preferably of about 5, 10, 50 or 100 mL.

In a third aspect, the invention provides for a method of treatmentcomprising the administration of a composition according to the firstaspect of the invention and/or the sequential or simultaneousadministration of a first and second compound of a kit of partsaccording to the second aspect of the invention.

Preferably, said method of treatment is a method for preventing,delaying and/or curing rosacea, more preferably treatment oftelangiectasia, erythema and/or flushing, preferably telangiectasia,erythema and/or flushing associated with inflammation inducedvasodilatation, as further detailed herein.

Encompassed in the invention is a composition according to the firstaspect of the invention and/or a kit of parts according to second aspectof the invention, for use as a medicament. Preferably, said compositionaccording to the first aspect of the invention and/or said kit of partsaccording to second aspect of the invention is for use in preventing,delaying and/or curing rosacea, more preferably for use in the treatmentof telangiectasia, erythema and/or flushing, preferably telangiectasia,erythema and/or flushing associated with inflammation inducedvasodilatation, as further detailed herein. Also encompassed in theinvention is the use of a composition according to the first aspect ofthe invention and/or a kit of parts according to second aspect of theinvention for the manufacture of a medicament. Preferably, saidmedicament is for preventing, delaying and/or curing rosacea, morepreferably for preventing, delaying and/or curing telangiectasia,erythema and/or flushing, preferably telangiectasia, erythema and/orflushing associated with inflammation induced vasodilatation, as furtherdetailed herein. Preferably, said composition according to the firstaspect, a first composition of a kit of parts according to the secondaspect and/or a second composition of a kit of parts according to thesecond aspect and/or medicament as defined herein is a topicalformulation understood herein as a formulation, including amicroencapsulated formulation, being suitable for topical administrationand may be in the form of a cream, ointment, solution, powder, spray,aerosol, capsule, solid or gel, and/or may be bonded to a solid surface,e.g. by immobilization with affinity ligands or throughionic/hydrophobic interactions and covalent immobilization.

A composition according to the first aspect of the invention and/or afirst and/or second composition of a kit of parts according to thesecond aspect of the invention may also form part of a body wash, soap,application stick or cosmetic.

A composition according to the first aspect and/or a second compositionof a kit of parts according to the second aspect and/or a mixtureresulting from admixing said first and second composition of a kit ofparts according to the second aspect just before administration asearlier indicated herein, is preferably said to be active, functional ortherapeutically active when it decreases the amount of bacterial cells,preferably gram positive bacterial cells, more preferably the amount ofStaphylococcus bacterial cells, most preferably the amount ofStaphylococcus aureus bacterial cells, present in a patient or in a cellof said patient or in a cell line or in a cell free in vitro system andpreferably means that 99%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%,5% or less of the initial amount of said bacterial cells is stilldetectable. More preferably, no bacterial cell, preferably no grampositive bacterial cell, more preferably no Staphylococcus bacterialcell, most preferably no Staphylococcus aureus bacterial cell, isdetectable. In this paragraph, the expression “amount of bacterialcells” preferably means viable bacterial cells. Staphylococci of allgenera may be detected using standard techniques known by the artisansuch as immunohistochemical techniques using Staphylococcus specificantibodies, tube coagulase tests that detect staphylocoagulase or “freecoagulase”, detection of surface proteins such as clumping factor (slidecoagulase test) and/or protein A (commercial latex tests). ViableStaphylococci may be detected using standard techniques known by theartisan such as microbiological bacterial culture techniques and/orreal-time quantitative reverse transcription polymerase chain reactionto assay for bacterial mRNA. A decrease in amount of bacterial cellsaccording to the invention is preferably assessed in a tissue or in acell of an individual or a patient by comparison to the amount presentin said individual or patient before treatment with said composition orpolypeptide of the invention. Alternatively, the comparison can be madewith a tissue or cell of said individual or patient which has not yetbeen treated with said composition or polypeptide in case the treatmentis local.

Preferably a composition according to the first aspect of the inventionand/or a second composition of a kit of parts of the second aspect ofthe invention, and/or a resulting mixture resulting from admixing thefirst and second composition of the kit of part of the second aspect ofthe invention just before administration as identified herein beforecomprises an amount of a second compound as defined herein which istherapeutically active as earlier identified herein. Preferably, saidcomposition is for topic administration to an individual in the needthereof, preferably to a patient suffering from rosacea, telangiectasia,erythema and/or flushing, and comprises said second compound in aneffective amount, preferably a concentration of 0.001-10% by weight ofthe total composition. Depending on the specific activity of the secondcompound, the effective amount may be as low about a few micrograms/mlsuch as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 microgram/ml to aboutseveral milligrams/ml such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10milligram/ml Preferably a composition according to the first aspect ofthe invention and/or a first composition of a kit of parts of the secondaspect of the invention, and/or a resulting mixture resulting fromadmixing the first and second composition of the kit of part of thesecond aspect of the invention just before administration as identifiedherein before is a composition for topical application for the treatmentof rosacea, telangiectasia, erythema and/or flushing, comprising avasoconstrictive compound according to the invention, selected from, butnot limited to a sympathomimetic compound according to the invention; ora combination of two or more of these. Preferably, said composition isfor topic administration to an individual in the need thereof,preferably to a patient suffering from rosacea, more preferablytelangiectasia, erythema and/or flushing, preferably telangiectasia,erythema and/or flushing associated with inflammation inducedvasodilatation, and comprises said vasoconstrictive compound accordingto the invention in an effective amount, preferably in the range of 0.01to 10% by weight of the total composition, preferably in the range of0.05 to 5%, more preferably in a range of 0.05 to 2.5%, even morepreferably in a range of 0.1 to 1%.

Preferably a composition according to the first aspect of the inventionand/or a first composition of a kit of parts of the second aspect of theinvention, and/or a resulting mixture resulting from admixing the firstand second composition of the kit of part of the second aspect of theinvention just before administration as identified herein before is acomposition for topic application for the treatment of rosacea, morepreferably telangiectasia, erythema and/or flushing, preferablytelangiectasia, erythema and/or flushing associated with inflammationinduced vasodilatation, comprising a vasoconstrictive compound accordingto the invention in the range of 0.05 to 5%, by weight of the totalcomposition preferably in a range of 0.05 to 2.5% more preferably in arange of 0.1 to 1%, even more preferably comprising about 0.25% of avasoconstrictive compound according to the invention. About is definedherein as a value minus or plus 10% of the indicated value.

A composition according to the first aspect, a first composition of akit of parts according to the second aspect and/or a second compositionof a kit of parts according to the second aspect and/or medicament asdefined herein may be in the liquid, solid or semi-liquid or semi-solidform, preferably further comprising a pharmaceutical acceptable carrier,excipient and/or stabilizer. Examples of pharmaceutically acceptablecarriers, excipients and stabilizers include, but are not limited to,buffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight polypeptides;proteins, such as serum albumin and gelatin; hydrophilic polymers suchas polyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as polysorbate (TWEEN™), polyethylene glycol (PEG), and poloxamers(PLURONICS™); and polymer thickeners such as hydrophilic andhydroalcoholic gelling agents frequently used in the cosmetic andpharmaceutical industries, preferably a gelling agent comprises betweenabout 0.2% and about 4% by weight composition. A composition accordingto the first aspect, a first composition of a kit of parts according tothe second aspect and/or a second composition of a kit of partsaccording to the second aspect and/or medicament as defined herein canalso include a lubricant, a wetting agent, a sweetener, a flavoringagent, an emulsifier, a suspending agent, a sun screen such as, but notlimited to, titanium dioxide or methyl cinnamate, and/or a preservative,in addition to the above ingredients, preferably a preservative ispresent as about 0.05% to 0.5% by weight of the total composition. Acomposition according to the first aspect, a first composition of a kitof parts according to the second aspect and/or a second composition of akit of parts according to the second aspect and/or medicament as definedherein can also include a carrier which are known in the art (such as acarbohydrate and a sugar-alcohol) to aid in the exposure of the skin toa medicament.

Preferably, a composition according to the first aspect, a firstcomposition of a kit of parts according to the second aspect and/or asecond composition of a kit of parts according to the second aspectand/or a medicament as defined herein further comprises and additionalactive ingredient. An additional active ingredient may be any of, but isnot limited to, a standard or conventional antibiotic agent such as, butnot limited to penicillin, synthetic penicillins, bacitracin,methicillin, cephalosporin, polymyxin, cefaclor, Cefadroxil, cefamandolenafate, cefazolin, cefixime, cefmetazole, cefonioid, cefoperazone,ceforanide, cefotanme, cefotaxime, cefotetan, cefoxitin, cefpodoximeproxetil, ceftazidime, ceftizoxime, ceftriaxone, cefriaxone moxalactam,cefuroxime, cephalexin, cephalosporin C, cephalosporin C sodium salt,cephalothin, cephalothin sodium salt, cephapirin, cephradine,cefuroximeaxetil, dihydratecephalothin, moxalactam, loracarbef mafateand/or chelating agents; an antifungal, such as, but not limited to,oxiconazole nitrate, ciclopirox olamine, ketoconazole, miconazolenitrate and butoconazole nitrate; an anti-androgen, such as, but notlimited to, flutamide and/or finasterid; a local anesthetic agent, suchas, but not limited to tetracaine, tetracaine hydrochloride, lidocaine,lidocaine hydrochloride, dyclonine, dyclonine hydrochloride,dimethisoquin hydrochloride, dibucaine, dibucaine hydrochloride,butambenpicrate and/or pramoxine hydrochloride; and dapsone which hasboth antimicrobial and anti-inflammatory properties. Other additionalactive ingredients may be selected from the groups listed in Culp andScheinfeld [8]. Another additional active ingredients may be ananti-inflammatory agent selected from the group consisting of acorticosteroid a calcineurin inhibitor, an immunotherapeutic compound, arecombinant human IFN-gamma, a microbial probiotic, a cytokinemodulator, an inflammatory cell recruitment blocker and a T cellactivation inhibitor. Preferred weight percentages of antimicrobialagents or other additional active agents are 0.1% to 10% weight of thetotal composition. Preferred weight percentages for local anestheticsare 0.025% to 5% by weight of the total composition.

A composition according to the first aspect, a first compositionaccording to a second aspect and/or a second composition according to asecond aspect and/or medicament as defined herein can be used to treatanimals, including humans, suffering from any of the conditions asidentified herein above, preferably from rosacea, more preferablytelangiectasia, erythema and/or flushing, preferably telangiectasia,erythema and/or flushing associated with inflammation inducedvasodilatation.

A preferred route of administration of said composition and/or saidmedicament is any suitable route of administration that can be used toadminister said composition according to the first aspect, said firstcomposition according to a second aspect and/or said second compositionaccording to a second aspect and/or medicament as defined hereinincluding but not limited to: oral, aerosol or other device for deliveryto the lungs, nasal spray, intravenous, intramuscular, intraperitoneal,intrathecal, vaginal, rectal, topical, lumbar puncture, intrathecal, anddirect application to the brain and/or meninges. Preferably, saidcomposition according to the first aspect, said first compositionaccording to a second aspect and/or said second composition according toa second aspect and/or medicament as defined herein are administeredtopical, preferably at the site of rosacea, more preferablytelangiectasia, erythema and/or flushing, preferably telangiectasia,erythema and/or flushing associated with inflammation inducedvasodilatation.

A preferred administration frequency of said composition and/or saidmedicament is once or twice a day, preferably to the area of the skinaffected. Preferably said treatment is continued as long as required forthe rosacea to be cleared. Preferably said treatment is continued for 2to 3 days, for 7 to 10 days and/or for 2 to 3 weeks. Preferably a totalamount of composition for topic application is administered asidentified herein resulting in a total application of about 1 gram ofvasoconstrictive compound to the person in the need thereof.

A preferred dosage of administration of said composition and/or saidmedicament is a dosage containing an effective total amount of saidfirst and second compound resulting in the prevention, delay and/or cureof rosacea, more preferably telangiectasia, erythema and/or flushing,preferably telangiectasia, erythema and/or flushing associated withinflammation induced vasodilatation.

Definitions

In the embodiments of the invention, telangiectasia, erythema and/orflushing, preferably telangiectasia, erythema and/or flushing associatedwith inflammation induced vasodilatation, are usually conditionsassociated with rosacea. These conditions (telangiectasia, erythemaand/or flushing, preferably telangiectasia, erythema and/or flushingassociated with inflammation induced vasodilatation) can however also bea phenomenon on its own, i.e. not necessarily associated with rosacea;treatment of these conditions not necessarily associated with rosacea isalso within the scope of the invention. The term “associated” herein inthe context of telangiectasia, erythema and/or flushing associated withinflammation induced vasodilatation” means that the telangiectasia,erythema and/or flushing could be caused by inflammation inducedvasodilatation but the telangiectasia, erythema and/or flushing couldalso be already present and be aggravated by inflammation inducedvasodilatation. In the embodiments of the invention, the inflammationinduced vasodilatation is preferably induced directly by a microbialcompound or antigen, or indirectly via innate and adaptive immunitymediated e.g. via a Toll-Like Receptors (TLR) or the complement system.A preferred TLR is TLR 2. Preferably the microbe is a Gram-positivebacterium, preferably a Staphylococcus, more preferably Staphylococcusaureus.

“Sequence identity” or “identity” in the context of amino acid- ornucleic acid-sequence is herein defined as a relationship between two ormore amino acid (peptide, polypeptide, or protein) sequences or two ormore nucleic acid (nucleotide, polynucleotide) sequences, as determinedby comparing the sequences. In the art, “identity” also means the degreeof sequence relatedness between amino acid or nucleotide sequences, asthe case may be, as determined by the match between strings of suchsequences. Within the invention, sequence identity with a particularsequence preferably means sequence identity over the entire length ofsaid particular polypeptide or polynucleotide sequence. The sequenceinformation as provided herein should not be so narrowly construed as torequire inclusion of erroneously identified bases. The skilled person iscapable of identifying such erroneously identified bases and knows howto correct for such errors. “Similarity” between two amino acidsequences is determined by comparing the amino acid sequence and itsconserved amino acid substitutes of one peptide or polypeptide to thesequence of a second peptide or polypeptide. In a preferred embodiment,identity or similarity is calculated over the whole SEQ ID NO asidentified herein. “Identity” and “similarity” can be readily calculatedby known methods, including but not limited to those described inComputational Molecular Biology, Lesk, A. M., ed., Oxford UniversityPress, New York, 1988; Biocomputing: Informatics and Genome Projects,Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis ofSequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., HumanaPress, New Jersey, 1994; Sequence Analysis in Molecular Biology, vonHeine, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov,M. and Devereux, J., eds., M Stockton Press, New York, 1991; andCarillo, H., and Lipman, D., SIAM J. Applied Math., 48:1073 (1988).Preferred methods to determine identity are designed to give the largestmatch between the sequences tested. Methods to determine identity andsimilarity are codified in publicly available computer programs.Preferred computer program methods to determine identity and similaritybetween two sequences include e.g. the GCG program package (Devereux,J., et al., Nucleic Acids Research 12 (1): 387 (1984)), BestFit, BLASTP,BLASTN, and FASTA (Altschul, S. F. et al., J. Mol. Biol. 215:403-410(1990). The BLAST X program is publicly available from NCBI and othersources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md.20894; Altschul, S., et al., J. Mol. Biol. 215:403-410 (1990). Thewell-known Smith Waterman algorithm may also be used to determineidentity.

Preferred parameters for polypeptide sequence comparison include thefollowing: Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453(1970); Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc.Natl. Acad. Sci. USA. 89:10915-10919 (1992); Gap Penalty: 12; and GapLength Penalty: 4. A program useful with these parameters is publiclyavailable as the “Ogap” program from Genetics Computer Group, located inMadison, Wis. The aforementioned parameters are the default parametersfor amino acid comparisons (along with no penalty for end gaps).Preferred parameters for nucleic acid comparison include the following:Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970);Comparison matrix: matches=+10, mismatch=0; Gap Penalty: 50; Gap LengthPenalty: 3. Available as the Gap program from Genetics Computer Group,located in Madison, Wis. Given above are the default parameters fornucleic acid comparisons.

Optionally, in determining the degree of amino acid similarity, theskilled person may also take into account so-called “conservative” aminoacid substitutions, as will be clear to the skilled person. Conservativeamino acid substitutions refer to the interchangeability of residueshaving similar side chains. For example, a group of amino acids havingaliphatic side chains is glycine, alanine, valine, leucine, andisoleucine; a group of amino acids having aliphatic-hydroxyl side chainsis serine and threonine; a group of amino acids having amide-containingside chains is asparagine and glutamine; a group of amino acids havingaromatic side chains is phenylalanine, tyrosine, and tryptophan; a groupof amino acids having basic side chains is lysine, arginine, andhistidine; and a group of amino acids having sulphur-containing sidechains is cysteine and methionine. Preferred conservative amino acidssubstitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine, andasparagine-glutamine. Substitutional variants of the amino acid sequencedisclosed herein are those in which at least one residue in thedisclosed sequences has been removed and a different residue inserted inits place. Preferably, the amino acid change is conservative. Preferredconservative substitutions for each of the naturally occurring aminoacids are as follows: Ala to ser; Arg to lys; Asn to gln or his; Asp toglu; Cys to ser or ala; Gln to asn; Glu to asp; Gly to pro; His to asnor gln; Ile to leu or val; Leu to ile or val; Lys to arg; gln or glu;Met to leu or ile; Phe to met, leu or tyr; Ser to thr; Thr to ser; Trpto tyr; Tyr to trp or phe; and, Val to ile or leu.

A polynucleotide is represented by a nucleotide sequence. A polypeptideis represented by an amino acid sequence. A nucleic acid construct isdefined as a polynucleotide which is isolated from a naturally occurringgene or which has been modified to contain segments of polynucleotideswhich are combined or juxtaposed in a manner which would not otherwiseexist in nature. Optionally, a polynucleotide present in a nucleic acidconstruct is operably linked to one or more control sequences, whichdirect the production or expression of said peptide or polypeptide in acell or in a subject.

As used herein the term “heterologous sequence” or “heterologous nucleicacid” is one that is not naturally found operably linked as neighboringsequence of said first nucleotide sequence. As used herein, the term“heterologous” may mean “recombinant”. “Recombinant” refers to a geneticentity distinct from that generally found in nature. As applied to anucleotide sequence or nucleic acid molecule, this means that saidnucleotide sequence or nucleic acid molecule is the product of variouscombinations of cloning, restriction and/or ligation steps, and otherprocedures that result in the production of a construct that is distinctfrom a sequence or molecule found in nature. “Operably linked” isdefined herein as a configuration in which a control sequence isappropriately placed at a position relative to the nucleotide sequencecoding for the polypeptide of the invention such that the controlsequence directs the production/expression of the peptide or polypeptideof the invention in a cell and/or in a subject.

“Operably linked” may also be used for defining a configuration in whicha sequence is appropriately placed at a position relative to anothersequence coding for a functional domain such that a chimeric polypeptideis encoded in a cell and/or in a subject.

Expression will be understood to include any step involved in theproduction of the peptide or polypeptide including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification and secretion.

Optionally, a promoter represented by a nucleotide sequence present in anucleic acid construct is operably linked to another nucleotide sequenceencoding a peptide or polypeptide as identified herein.

The term “transformation” refers to a permanent or transient geneticchange induced in a cell following the incorporation of new DNA (i.e.DNA exogenous to the cell). When the cell is a bacterial cell, as isintended in the current invention, the term usually refers to anextrachromosomal, self-replicating vector which harbors a selectableantibiotic resistance.

An expression vector may be any vector which can be convenientlysubjected to recombinant DNA procedures and can bring about theexpression of a nucleotide sequence encoding a polypeptide of theinvention in a cell and/or in a subject. As used herein, the term“promoter” refers to a nucleic acid fragment that functions to controlthe transcription of one or more genes or nucleic acids, locatedupstream with respect to the direction of transcription of thetranscription initiation site of the gene. It is related to the bindingsite identified by the presence of a binding site for DNA-dependent RNApolymerase, transcription initiation sites, and any other DNA sequences,including, but not limited to, transcription factor binding sites,repressor and activator protein binding sites, and any other sequencesof nucleotides known to one skilled in the art to act directly orindirectly to regulate the amount of transcription from the promoter.Within the context of the invention, a promoter preferably ends atnucleotide-1 of the transcription start site (TSS).

“Polypeptide” as used herein refers to any peptide, oligopeptide,polypeptide, gene product, expression product, or protein. A polypeptideis comprised of consecutive amino acids. The term “polypeptide”encompasses naturally occurring or synthetic molecules. The term“control sequences” is defined herein to include all components, whichare necessary or advantageous for the expression of a polypeptide. Eachcontrol sequence may be native or foreign to the nucleic acid sequenceencoding the polypeptide. Such control sequences include, but are notlimited to, a leader, optimal translation initiation sequences (asdescribed in Kozak, 1991, J. Biol. Chem. 266:19867-19870), apolyadenylation sequence, a pro-peptide sequence, a pre-pro-peptidesequence, a promoter, a signal sequence, and a transcription terminator.At a minimum, the control sequences include a promoter, andtranscriptional and translational stop signals.

The control sequences may be provided with linkers for the purpose ofintroducing specific restriction sites facilitating ligation of thecontrol sequences with the coding region of the nucleic acid sequenceencoding a polypeptide.

The control sequence may be an appropriate promoter sequence, a nucleicacid sequence, which is recognized by a host cell for expression of thenucleic acid sequence. The promoter sequence contains transcriptionalcontrol sequences, which mediate the expression of the polypeptide. Thepromoter may be any nucleic acid sequence, which shows transcriptionalactivity in the cell including mutant, truncated, and hybrid promoters,and may be obtained from genes encoding extracellular or intracellularpolypeptides either homologous or heterologous to the cell.

The control sequence may also be a suitable transcription terminatorsequence, a sequence recognized by a host cell to terminatetranscription. The terminator sequence is operably linked to the 3′terminus of the nucleic acid sequence encoding the polypeptide. Anyterminator, which is functional in the cell, may be used in theinvention.

The control sequence may also be a suitable leader sequence, anon-translated region of a mRNA which is important for translation bythe host cell. The leader sequence is operably linked to the 5′ terminusof the nucleic acid sequence encoding the polypeptide. Any leadersequence, which is functional in the cell, may be used in the invention.

The control sequence may also be a polyadenylation sequence, a sequencewhich is operably linked to the 3′ terminus of the nucleic acid sequenceand which, when transcribed, is recognized by the host cell as a signalto add polyadenosine residues to transcribed mRNA. Any polyadenylationsequence, which is functional in the cell, may be used in the invention.

In this document and in its claims, the verb “to comprise” and itsconjugations is used in its non-limiting sense to mean that itemsfollowing the word are included, but items not specifically mentionedare not excluded. In addition the verb “to consist” may be replaced by“to consist essentially of” meaning that a product or a composition or anucleic acid molecule or a peptide or polypeptide of a nucleic acidconstruct or vector or cell as defined herein may comprise additionalcomponent(s) than the ones specifically identified; said additionalcomponent(s) not altering the unique characteristic of the invention. Inaddition, reference to an element by the indefinite article “a” or “an”does not exclude the possibility that more than one of the elements ispresent, unless the context clearly requires that there be one and onlyone of the elements. The indefinite article “a” or “an” thus usuallymeans “at least one”.

All patent and literature references cited in the present specificationare hereby incorporated by reference in their entirety.

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the invention in any way.

EXAMPLES Example 1: Treatment of Rosacea Patients

Rosacea patients are treated with a combination of an antibacterialagent and a vasoconstrictor according to the invention. The treatmentresults in effective treatment of rosacea, telangiectasia, erythemaand/or flushing; the bacterial trigger for inflammation is suppressed bythe antibacterial agent, resulting in less inflammation-relatedvasodilatation, in turn allowing a lower need for a vasoconstrictivecompound.

Example 2: Combination Treatment with a Vasoconstrictor and anAntibacterial Specific for Staphylococcus aureus; a Comparative Study

In several forms of dermatitis, like rosacea, courses of topicalvasoconstrictor therapy are used to suppress symptoms of local redness.However, vasoconstrictors such as brimonidine do not treat theunderlying cause of the vasodilatation causing the redness, and symptomsare known to return quickly after the alfa-adrenergic vasoconstrictiveeffect has waned.

The inventors considered that of the underlying vasodilatory triggersfor local redness is inflammation, that can be caused by localirritation or infection of the skin by bacteria, like Staphylococcusaureus. Staphylococcus aureus is the most prevalent cause of bacterialskin infections and is considered to play a role in inflammatory skinconditions like eczema and acne as a local trigger of inflammatorysymptoms and (secondary) infection.

The inventors hypothesized that a specific antibacterial compound wouldbe effective in combination with vasoconstriction treatment ininflammatory skin conditions like rosacea. Accordingly, a compoundspecifically targeting Staphylococcus aureus was used. The compoundStaphefekt™ SA. 100 (comprised in Gladskin™) was obtained from MicreosHuman Health B.V., The Netherlands. Since Staphefekt™ eradicates S.aureus as an etiological factor of local inflammation, it washypothesized that Staphefekt™ treatment combined with vasoconstrictortreatment would alleviate the total burden of symptoms in inflammatoryskin conditions like rosacea, comprising amongst others inflammatory andvascular symptoms, in a synergistic manner, as both the inflammatorytrigger, causing vasodilatation amongst other symptoms, and thevasodilatation itself was targeted.

To study the use of vasoconstrictor treatment during Staphefekt™treatment, the relative reduction in total symptom score was comparedbetween a study conducted with Staphefekt™ (Gladskin™) in a cohort ofrosacea patients, and a case study of rosacea first using monotherapywith a vasoconstrictor (Mirvaso™ gel, comprising 0.3% w/w brimonidine)and thereafter using combination therapy of the vasoconstrictor withStaphefekt™ (Gladskin™).

Surprisingly, the reduction in total symptom scores with the combinationof the vasoconstrictor in combination with Staphefekt™ was considerablylarger than the sum of the reductions achieved with Staphefekt™ alone orthe vasoconstrictor alone, evidencing the synergistic effect ofcombination treatment using both compounds on vascular symptoms ininflammatory skin diseases (FIG. 1).

The synergistic effect of the combination of a vasoconstrictor andStaphefekt™ also implies that a lower dose or shorter course ofvasoconstrictor treatment would be effective in combination therapy withStaphefekt™ to achieve the same amount of total symptom reduction.

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TABLE 1 SEQ ID NO overview table SEQ ID NO Construct Organism 1 Ply2638endolysin CDS Bacteriophage 2638A 2 Ply2638 endolysin PRT Bacteriophage2638A 3 CWT-LST CDS S. simulans 4 CWT-LST PRT S. simulans 5 CBD2638 CDSBacteriophage 2638A 6 CBD2638 PRT Bacteriophage 2638A 7 CWT-NM3 CDS S.aureus phage phiNM3 8 CWT-NM3 PRT S. aureus phage phiNM3 9 CHAPK CDS S.phage K 10 CHAPK PRT S. phage K 11 CHAP-ΦTwort CDS S. phage Twort 12CHAP-ΦTwort PRT S. phage Twort 13 M23-2638 CDS Bacteriophage 2638A 14M23-2638 PRT Bacteriophage 2638A 15 M23-LST CDS S. simulans 16 M23-LSTPRT S. simulans 17 Ami2638 CDS Bacteriophage 2638A 18 Ami2638 PRTBacteriophage 2638A 19 CHAPK_CHAPK_CWT-LST CDS artificial construct 20CHAPK_CHAPK_CWT-LST PRT artificial construct 21 M23-LST_M23-LST_CWT-LSTCDS artificial construct 22 M23-LST_M23-LST_CWT-LST PRT artificialconstruct 23 Ami2638_ami2638_CWT-LST CDS artificial construct 24Ami2638_ami2638_CWT-LST PRT artificial construct 25 HXaAmi2638_CBD2638CDS artificial construct 26 HXaAmi2638_CBD2638 PRT artificial construct27 HXaAmi2638_CWT-LST CDS artificial construct 28 HXaAmi2638_CWT-LST PRTartificial construct 29 HXaAmi2638_CWT-NM3 CDS artificial construct 30HXaAmi2638_CWT-NM3 PRT artificial construct 31 HXaCHAPK_CBD2638 CDSartificial construct 32 HXaCHAPK_CBD2638 PRT artificial construct 33HXaCHAPK_CWT-LST CDS artificial construct 34 HXaCHAPK_CWT-LST PRTartificial construct 35 HXaCHAPK_CWT-NM3 CDS artificial construct 36HXaCHAPK_CWT-NM3 PRT artificial construct 37 HXaCHAPTw_CBD2638 CDSartificial construct 38 HXaCHAPTw_CBD2638 PRT artificial construct 39HXaCHAPTw_CWT-LST CDS artificial construct 40 HXaCHAPTw_CWT-LST PRTartificial construct 41 HXaCHAPTw_CWT-NM3 CDS artificial construct 42HXaCHAPTw_CWT-NM3 PRT artificial construct 43 HXaM23-LST_CBD2638 CDSartificial construct 44 HXaM23-LST_CBD2638 PRT artificial construct 45HXaM23-LST_CWT-LST CDS artificial construct 46 HXaM23-LST_CWT-LST PRTartificial construct 47 HXaM23-LST_CWT-NM3 CDS artificial construct 48HXaM23-LST_CWT-NM3 PRT artificial construct 49HXaAmi2638_Ami2638_CBD2638 CDS artificial construct 50HXaAmi2638_Ami2638_CBD2638 PRT artificial construct 51HXaAmi2638_Ami2638_CWT-LST CDS artificial construct 52HXaAmi2638_Ami2638_CWT-LST PRT artificial construct 53HXaAmi2638_Ami2638_CWT-NM3 CDS artificial construct 54HXaAmi2638_Ami2638_CWT-NM3 PRT artificial construct 55HXaCHAPK_CHAPK_CBD2638 CDS artificial construct 56HXaCHAPK_CHAPK_CBD2638 PRT artificial construct 57HXaCHAPK_CHAPK_CWT-LST CDS artificial construct 58HXaCHAPK_CHAPK_CWT-LST PRT artificial construct 59HXaCHAPK_CHAPK_CWT-NM3 CDS artificial construct 60HXaCHAPK_CHAPK_CWT-NM3 PRT artificial construct 61HXaCHAPTw_CHAPTw_CBD2638 CDS artificial construct 62HXaCHAPTw_CHAPTw_CBD2638 PRT artificial construct 63HXaCHAPTw_CHAPTw_CWT-LST CDS artificial construct 64HXaCHAPTw_CHAPTw_CWT-LST PRT artificial construct 65HXaCHAPTw_CHAPTw_CWT-NM3 CDS artificial construct 66HXaCHAPTw_CHAPTw_CWT-NM3 PRT artificial construct 67HXaM23-LST_M23-LST_CBD2638 CDS artificial construct 68HXaM23-LST_M23-LST_CBD2638 PRT artificial construct 69HXaM23-LST_M23-LST_CWT-LST CDS artificial construct 70HXaM23-LST_M23-LST_CWT-LST PRT artificial construct 71HXaM23-LST_M23-LST_CWT-NM3 CDS artificial construct 72HXaM23-LST_M23-LST_CWT-NM3 PRT artificial construct 73 His-tag withlinker CDS artificial construct 74 His-tag with linker PRT artificialconstruct 75 LST CDS S. simulans 76 LST PRT S. simulans 77HXaCHAP11_M23-2638_Ami2638_CBD2638 CDS artificial construct 78HXaCHAP11_M23-2638_Ami2638_CBD2638 PRT artificial construct 79HXaAmi11_M23-2638_Ami2638_CBD2638 CDS artificial construct 80HXaAmi11_M23-2638_Ami2638_CBD2638 PRT artificial construct 81HXaCHAPTw_Ami2638_M23-LST_CBD2638 CDS artificial construct 82HXaCHAPTw_Ami2638_M23-LST_CBD2638 PRT artificial construct 83HXaM23-LST_Ami2638_CBD2638 CDS artificial construct 84HXaM23-LST_Ami2638_CBD2638 PRT artificial construct 85HXaM23-2638_Ami2638_CBD2638_CBD2638 CDS artificial construct 86HXaM23-2638_Ami2638_CBD2638_CBD2638 PRT artificial construct 87HXaM23-2638_CBD2638 CDS artificial construct 88 HXaM23-2638_CBD2638 PRTartificial construct 89 HXaPly2638-Ply2638 CDS artificial construct 90HXaPly2638-Ply2638 PRT artificial construct 91HXaCHAPTw_Ami2638_M23-LST_CWT-LST CDS artificial construct 92HXaCHAPTw_Ami2638_M23-LST_CWT-LST PRT artificial construct 93 HXaLST_LSTCDS artificial construct 94 HXaLST_LST PRT artificial construct 95HXaGFP_CBD2638 CDS artificial construct 96 HXaGFP_CBD2638 PRT artificialconstruct 97 CHAPΦ11 CDS S. aureus phage phi 11 98 CHAPΦ11 PRT S. aureusphage phi 11 99 AmiΦ11 CDS S. aureus phage phi 11 100 AmiΦ11 PRT S.aureus phage phi 11 101 EP15158880.3 endolysin PRT artificial construct

The invention claimed is:
 1. A method for treating rosacea in a patientin need thereof comprising the sequential or simultaneous administrationof a first compound and a second compound to the patient, wherein saidfirst compound is a vasoconstrictive compound selected from the groupconsisting of alpha(1)- and alpha(2)-adrenergic agonists and said secondcompound is a compound specifically targeting a Staphylococcus cell, andcomprises one or more enzymatic active domains exhibiting peptidoglycanhydrolase activity.
 2. The method according to claim 1, wherein saidsecond compound further comprises at least one cell wall binding domainwhich specifically binds to the peptidoglycan cell wall of saidStaphylococcus cell.
 3. The method according to claim 2, wherein saidcell wall binding domain originates from or is a homologue of aStaphylococcus phage endolysin, an S. simulans lysostaphin and/or an S.capitis ALE-1 bacteriocin.
 4. The method according to claim 2, whereinsaid second compound comprises a cell wall binding domain that has atleast 80% identity to SEQ ID NO:
 6. 5. The method according to claim 1,wherein said one or more enzymatic active domains is selected from thegroup consisting of a cysteine, histidine dependentamidohydrolase/peptidase domain, an endopeptidase domain, an amidasedomain and a glycosylhydrolase domain, and combinations thereof.
 6. Themethod according to claim 1, wherein said second compound is a naturallyoccurring endolysin.
 7. The method according to claim 1, wherein saidsecond compound is a recombinant polypeptide comprising the one or moreenzymatic active domains exhibiting peptidoglycan hydrolase activity. 8.The method according to claim 1, wherein the alpha(1)- andalpha(2)-adrenergic agonist is selected from the group consisting ofBrimonidine, Tetrahydrozoline and Oxymetazoline.